The Ubiquitin System Plays A Role In Synaptic Plasticity And Memory In Aplysia

decrease in the overall ratio of regulatory to catalytic subunits, promoting an excess of free, active catalytic subunits and a persistent increase in PKA activity. By this clever mechanism, a chain of events is set in motion whereby a biochemical effect is established in the cell that outlasts the initial, triggering elevation of the second messenger cAMP. The PKA will remain activated until compensatory resynthesis of new regulatory subunit occurs, or until the catalytic subunit is degraded. Interestingly, although the mechanism has not yet been worked out, recent evidence indicates that the DAG-responsive effector PKC also is persistently activated after serotonin stimulation of sensory neurons. Available evidence indicates that the persistent activation of PKA underlies an intermediate stage of facilitation, lasting on the order of many hours after the triggering applications of serotonin are finished. Interestingly, pioneering work on this mechanism was performed using sensiti-zation training in animals, emphasizing the strong likelihood of this mechanism contributing to the underlying cellular basis for the change in the animal's behavior. We will return to the Aplysia system in greater detail in Chapter 12. For the present, it represents an interesting example of regulation of synaptic plasticity by the ubiquitin system, and thus an interesting parallel to the identification of a role for ubiquitination in memory based on studies of Angelman Syndrome.

To the extent that deficiencies in the mouse model for Angelman Syndrome reflect those in human Angelman Syndrome patients, Yong-hui's data suggest that defects in hippocampal long-term poten-tiation may underlie the learning defects exhibited in Angelman Syndrome. Angelman mice have normal synaptic transmission, short-term plasticity, and hippocampal morphology. Anatomical studies of humans indicate that their hippocampal morphology is similarly normal. Thus, Angelman Syndrome humans appear to have a selective deficit in synaptic plasticity with normal baseline function based on their anatomical characterization and extrapolating from the mouse model findings. Because the UBE3A gene is imprinted, AS patients likely have this loss of synaptic plasticity restricted to their hippocampus (and cerebellum). Thus, these patients appear to have a very precise and selective lesion, in contrast to patients such as H. M. where there is extensive and imprecise anatomical derangement and, of course, loss of anatomical connections to and from the hippocampus. To the best of our ability to determine what is happening in the human based on studies of the mouse model, selective deficits in hippocampal long-term synaptic plasticity are what lead to the profound learning and memory deficits of Angelman Syndrome. In my mind, this is one of the most important findings to come out of this work.

What are the targets of the E6-AP ubiq-uitin ligase pathway that lead to this striking memory dysfunction? One possibility, as mentioned previously, is that this E3 ligase is serving as a signal transduction pathway that modulates downstream targets acutely. Although this is a possibility, there is no direct evidence to suggest this at present, and a much more parsimonious hypothesis is that the E6-AP is playing the more traditional role of controlling the level of target proteins by sending them to the proteasome. In this scenario, loss of E6-AP will secondarily lead to elevations in the levels of downstream targets resulting from loss of this mechanism for their degradation. At present, we prefer this hypothesis of secondary effects to increase the levels of downstream targets, although it is certainly always necessary to keep an open mind.

In considering this hypothesis, I note that it does not immediately lead to a lot of specific possibilities. The E6AP E3 ligase apparently has a very restricted set of substrates, as mentioned earlier, so not many candidates come to mind as downstream targets. One known substrate is the p53 tumor suppressor. In fact, maternal deficiency mice exhibit altered levels of p53 in hippocampal pyramidal neurons, so this is one potential mechanism. Disappointingly, however, p53 function is difficult to tie in to synaptic plasticity based on our present state of knowledge. In short, nothing specific about the function of p53 suggests how it could lead to a derangement of LTP and memory.

To try to address the problem in a different way, Ed Weeber in my lab decided to test for derangements in the signal transduction mechanisms that we already knew were involved in normal memory formation. In collaboration with Yong-hui and Art Beaudet, Ed set about using hippocampal tissue from the Angelman mouse model in Western blotting screens, to see if any candidate molecules know to function in LTP could be identified. To make a long story very short, after looking at a wide variety of specific proteins and protein kinases, we found an alteration in hippocampal CaMKII.

The alteration was in Thr286 autophos-phorylation, not total protein level, which was quite surprising to us because we had expected the loss of proteolysis of a target protein to lead to an increase in the level of that protein. Nevertheless, Ed observed that hippocampal extracts from Angelman mice exhibited a selective increase in Thr286 autophosphorylation with no change in total protein.

Based on the known property of Thr286 autophosphorylation to render the kinase autonomously active, we next tested these hippocampal extracts for increases in CaMKII activity. In fact, to our further surprise in these studies, we found that Angelman mice had decreased CaMKII activity. Further studies in collaboration with Ype Elgersma and Alcino Silva indicated the answer to this mystery. These studies showed that there was aberrant hyperphosphorylation of CaMKII at the inhibitory site, thr305/306. This increased inhibitory autophosphorylation is the likely mechanism through which there is a diminution of CaMKII activity in Angelman hippocampus.

Is this single molecular change, increased autophosphorylation at thr305, sufficient to cause Angelman Syndrome? In a complementary series of studies, Alcino's lab converged on this same hypothesis using a transgenic point mutant animal that mimicked hyperphosphorylation at the 305 site, a transgenic animal expressing a thr-to-asp CaMKII point mutant. Their motivation for generating this animal arose from their years of work investigating the details of CaMKII function and phosphorylation in synaptic plasticity and memory. Strikingly, the data from Alcino's lab indicated that the 305 hyperphos-phorylation is indeed sufficient to give an LTP and learning phenotype reminiscent of Angelman syndrome. Thus, approaching a problem from two very different viewpoints, my lab and Alcino's once again converged on a common answer.

We do not know the basis of the hyper-phosphorylation. Our current working hypothesis, which is very speculative, is that E6AP regulates the level of some protein(s) that controls phosphatase activity, perhaps a phosphatase inhibitor. This would link up proteolysis, which presumably controls the steady-state level of some protein in the cell, with the phosphoryla-tion increase that we have observed. As mentioned earlier, we certainly at this point cannot rule out the involvement of a more acute, signal—transduction type process wherein ubiquitination acutely controls a target's catalytic activity.

Overall these data strongly indicate that the normal function of the CaMKII cascade is necessary for human synaptic plasticity and memory. Specifically, these findings implicate the subtle derangement of regulatory mechanisms for CaMKII in the pronounced memory dysfunction of Angelman Syndrome. As was the case with studies of neurofibromatosis and the ras/ERK cascade, once again decades of study of the basic signal transduction mechanisms for synaptic plasticity and memory converged with studies of human mental retardation.

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